Work on the role of insulin-like growth factors (IGFs) in pediatric sarcomas is continuing to focus on potential therapeutic approaches as well as further defining the downstream signaling events of IGF action. We have developed a CRADA with Immunogen to test a new humanized monoclonal antibody directed against the IGFI receptor. The antibody is highly specific with a Kd in the low nanomolar range. Studies are ongoing testing its effect in an orthotopic model of rhabdomyosarcoma that is driven by an autocrine IGFII signaling pathway. We are evaluating direct effects of the antibody on vivo on phosphorylation and activation of IGF signaling components. We have completed studies demonstrating that liver IGF-I deficient mice (LID mice) have decreased spontaneous metastases in an osteosarcoma model. This model involved bone marrow transplant, so we are currently working to develop the LID phenotype in a Balb/c mouse background to allow further studies on the role of IGF-I in metastatic behavior. We recently reported that in rhabdomyosarcoma and a mouse skeletal muscle cell line that PTEN specifically modulates AKT phosphorylation on only 1 of 2 major phosphorylation sites. These observations should help design specific inhibitors of this pathway. We have continued to study the role of mTOR signaling in pediatric sarcomas. We have recently linked ezrin expression and metastatic potential (see below) to mTOR activation and have reported that mTOR blockade using CCI 779 in rhabdomyosarcomas inhibits primary tumor growth and inhibits mTOR signaling in the tumors. Ongoing studies are determining the effect of CCI 779 on metastatic behavior. We have continued our studies on the molecular determinants of metastases, and recently reported that ezrin expression is strongly associated with metastatic behavior in both our Balb/c model of osteosarcoma, and a genetic model of rhabdomyosarcoma. In both tumors, we have now reported that reduction of ezrin using antisense or RNAi leads to dramatically decreased metastatic behavior. As noted above, we have now linked ezrin signaling to activation of mTOR, and thus are exploring mTOR inhibitors as potential anti-metastatic agents.